The herbicide research strategy targets to develop new herbicide tolerant crop (HTC) traits. Three main strategies are available for making plants tolerant to herbicides, i.e. (1) detoxifying the herbicide with an enzyme which transforms the herbicide, or its active metabolite, into non-toxic products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP242236, EP337899); (2) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide, or to its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP293356, Padgette S. R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide, in view of the kinetic constants of this enzyme, so as to have enough of the functional enzyme available despite the presence of its inhibitor.
However, identifying new target enzymes which confer herbicide tolerance to a plant when over-expressed and/or mutated is often time consuming due to the long regeneration period of plants upon genetic manipulation and exposure to the test herbicide. The problem of the present invention, thus, resides in the provision of a rapid assay by means of which a selection of polynucleotides can be rapidly screened for their capacity or efficiency to confer herbicide tolerance in plants. The inventors of the present invention have solved this problem by combining the transient expression of candidate genes in plant cells with an assay for measuring chlorophyll fluorescence of plant cells that have been subjected to a certain stress condition, i.e. treatment of the cells with an herbicidal compound.
Chlorophyll fluorescence is light reemitted after being absorbed by chlorophyll molecules of plant leaves. Light energy that has been absorbed by a leaf will excite electrons in chlorophyll molecules. Energy in photosystem II can be converted to chemical energy to drive photosynthesis (photochemistry). If photochemistry is inefficient, excess energy can damage the leaf. Energy can be emitted (known as energy quenching) in the form of heat (called non-photochemical quenching NPQ) or emitted as chlorophyll fluorescence. These three processes are in competition, so fluorescence yield is high when less energy is emitted as heat or used in photochemistry. Therefore, by measuring the amount of chlorophyll fluorescence, the efficiency of photochemistry and non-photochemical quenching can be assessed. The fluorescence emitted from a leaf has a longer wavelength than the light absorbed by the leaf. Therefore, fluorescence can be measured by shining a defined wavelength of light onto a leaf and measuring the level of light emitted at longer wavelengths. According to Gitelson et al., the ratio between chlorophyll fluorescence at 735 nm and the wavelength range 700 nm to 710 nm, F735/F700 could be used as a precise indicator of chlorophyll content in plant leaves [Gitelson, et al (1999). Remote Sensing of Environment 69 (3): page 296]. Fluorescence is induced by direct excitation of chlorophyll molecules of photosystem II (PSII) by light and their immediate relaxation. The chloroplast fluorescence results from the reactions of deexcitation of excited chlorophyll molecules. Under ideal conditions, most of the energy from excited molecules is trapped into chemical energy which reduces the fluorescence yield often designated as chlorophyll fluorescence quenching. The amount and degree of variable fluorescence is a measure of chloroplast activity (Mir, N. A., et al., Plant Physiol 108:313-318 (1995)). When PSII is functioning poorly, fluorescence characteristics are altered. Stress exposures such as chilling injury (van Kooten, O., and Snell, Photosyn. Res. 25:147-150 (1990)) and high temperature stress (Havaux, M., et al., Planta 186:88-89 (1991)) can be detected as a reduction in PSII function.
Li et al., 2008, (J. Integr. Plant Biol. doi: 10.1111/j.1744-7909.2008.00686.x) reported that chlorophyll a fluorescence imaging system has become ubiquitous in plant ecophysiology studies (Maxwell and Johnson 2000, J. Exp. Bot. 51, 659-668). As the measurement is nondestructive, rapid and convenient, chlorophyll fluorescence method has many advantages in the quantification of stress effects on photosynthesis (Krause and Weis 1991, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 313-349). Based on pulse amplitude modulation (PAM) and the saturation pulse method (Schreiber et al. 1986, Photosynth. Res. 10, 51-62), chlorophyll fluorescence yield provides quantitative information not only on steady-state photosynthesis, but also on various mechanisms of protection against stress-induced damage by excess radiation (Govindjee 1995, Aust. J. Plant Physiol. 22, 131-160; Demmig-Adams and Adams 1996, Trends Plant Sci. 1, 21-26; Kramer and Crofts 1996 Photosynth. Environ. 5, 25-66; Meng et al. 2001, New Phytol. 151, 585-595).
Dayan and Zaccaro (Pesticide Biochemistry and Physiology 2012, 103, 189-197) have developed a three-step assay to test selected herbicides and to determine whether induced chlorophyll fluorescence is a suitable marker to identify certain herbicide modes of action.